Pharmaceutical composition comprising erythrocytes encapsulating a PLP-dependent enzyme and, a non-phosphate PLP precursor

ABSTRACT

The invention relates to a pharmaceutical composition containing a PLP-dependent enzyme and optionally its cofactor, pyridoxal phosphate (PLP), and/or a phosphate or non-phosphate precursor of PLP, its use as a drug, its production method and a therapeutic treatment method related to it. The pharmaceutical composition comprises erythrocytes and a pharmaceutically acceptable vehicle, the erythrocytes encapsulating the PLP-dependent enzyme. The PLP-dependent enzyme may be methioninase, tyrosine phenol-lyase, tyrosine aminotransferase or cystathionine beta-synthase.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 16/102,171 filedAug. 13, 2018 and issued as U.S. Pat. No. 10,780,126 on Sep. 22, 2020,which was a continuation of U.S. Ser. No. 15/117,588 having a filingdate of Aug. 9, 2016 and issued as U.S. Pat. No. 10,046,009 on Aug. 14,2018, which was a 371 application of International applicationPCT/EP2015/052962, filed on Feb. 12, 2015, which claimed the benefit ofFrench Patent Application, FR 14 51100, filed on Feb. 12, 2014, all ofsaid applications incorporated herein by reference.

The invention relates to a pharmaceutical composition containing aPLP-dependent enzyme, for example methioninase, and optionally itscofactor, pyridoxal phosphate (PLP or P5P), and/or a phosphate ornon-phosphate precursor of PLP, to its use as a drug, to its productionmethod and to a therapeutic treatment method relating to it.

Pyridoxal phosphate (PLP), a derivative of vitamin B6, is a cofactorused for a large variety of enzymes. Called herein “PLP enzymes” forPLP-dependent enzymes, they form a group of about 145 distinct enzymesinvolved for most of them in metabolic pathways for transforming aminoacids. The reaction catalyzed by these enzymes includesdecarboxylations, transaminations or further removal reactions(Percudani and Perrachi, EMBO reports Vol. 4 no. 9, 2003).

Because of the large number of enzymes belonging to the group of PLPenzymes and of reactions catalyzed by the latter, their potential use inhuman therapeutics has been investigated. From among the differentopportunities for therapeutic intervention associated with PLP enzymes,their use in the treatment of cancer and of cardiovascular pathologieshas been the subject of many studies (EI-Sayed and Shindia “Targets ingene therapy” Prof. Yongping You ed., 2011). More particularly,methioninase would be of interest for depleting plasma methionine andinducing apoptosis of auxotrophic tumoral cells for this amino acid. Itwas shown that many human tumoral cells were incapable of proliferatingwhen methionine is replaced with its precursor homocysteine while normalcells have the capability of proliferating in such a medium. Thisdependency on methionine was notably observed for cell lines derivedfrom breast, lung, colon, kidney, bladder, melanoma and glioblastomacancers (Durando et al., Bull Cancer 2008; 95 (1): 69-76).

In spite of the therapeutic interest of the PLP enzymes, development oftreatment based on an administration via a systemic route of theseenzymes comes up against significant limitations:

-   -   the PLP enzymes are mainly obtained from prokaryotic organisms        and are therefore strongly immunogenic in the case of        administration to humans    -   their half-life in plasma is short, requiring resorting to        frequent administrations or to large doses in order to be able        to obtain sufficient activity    -   the low bioavailability of the PLP cofactor in plasma causes a        rapid drop of their activity after administration.

These limitations were widely described in the case of methioninase. Sunet al. have produced a recombinant methioninase in the Escherichia colibacterium from the gene coding for the enzyme extracted from thePseudomonas putida bacterium. The thereby obtained enzyme called rMETasewas injected intravenously to immunodeficient mice. Twenty four hoursafter injection, the plasma activity of the enzyme, determined in vitrowithout adding PLP, was undetectable, indicating its short action period(Sun et al. Cancer Research 63, 8377-8383, 2003).

A year later, the same team published the results of administration ofrMETase in macaques (Yang et al. Clinical Cancer Research Vol. 10,2131-2138, 2004). In this study, rMETase doses of 1,000, 2,000 and 4,000units/kg were administered intravenously to six monkeys. A secondinjection was performed 28 days after the first and caused in twomonkeys an anaphylactic shock causing the death of one of the twoanimals. The immunogenicity of rMETase moreover caused the developmentof anti-rMETase antibodies of the IgG type (a majority) and of the IgMtype for the majority of the treated animals (four out of six). Theneutralizing nature of these antibodies was demonstrated in vitro.

In order to overcome the short half-life and immunogenicity limitationsof their methioninase, the same authors then proposed resorting topegylation of their enzyme. The grafting of PEG groups is a knowntechnique for increasing the half-life and reducing the immunogenicityof therapeutic proteins. Activated PEG derivatives were put into thepresence of rMETase in order to obtain PEG-rMETase. This modification ofthe enzyme caused an increase in the half-life in mice from 2 h for thefree enzyme to 38 h for the PEG-rMETase. This significant increase inhalf-life is accompanied with a reduction in immunogenicity (Sun et al.Cancer Research 63, 8377-8383, 2003).

If pegylation partly met the problems of half-life and immunogenicity, amajor problem of PLP enzymes remains: the low bioavailability of thecofactor in the plasma. The PLP enzymes are catalytically active in thepresence of their cofactor, PLP, this is then referred to as aholoenzyme. After injection, the holoenzyme is rapidly converted into aninactive apoenzyme because of the loss of the PLP cofactor.

PLP brought in an exogenous way is rapidly unavailable for the enzyme,the plasma half-life of the free PLP only being about 15 minutes. Thisphenomenon was demonstrated in the case of the PLP enzyme, tyrosinephenol-lyase (TPL). Elmer et al. (Cancer Research 38; 3663-3667, 1978)purified TPL and injected it into normal mice. Five hours after theinjection, blood samples were taken in order to assay the activity ofthe TPL. This activity assay was carried out according to twoconditions: a portion of the samples were assayed without adding PLP,the other portion was assayed with addition of an optimal amount of PLPin the reaction mixture for the assay (both of these conditionsreflecting the actually measured activity in plasma and the potentialactivity of the enzyme if it had access to its PLP cofactor). Thecomparison of the obtained results shows that only 7% of the potentialactivity of the TPL is actually measured in the plasma. The same testwas conducted with a group of mice, wherein, concomitantly to theinjection of TPL, a large amount of PLP was administered, and thenre-injections of PLP were carried out every hour. In this scenario, thecomparison of the assay results shows that 37% of the potential activityis actually measured in the plasma. Co-administration of PLP thereforegave the possibility of improving in a limited way the activity of TPLin plasma. However, PLP provided in an exogenous way is rapidlyunavailable for TPL, the plasma half-life of free PLP being of about 15minutes. Therefore, the rise in the plasma level of PLP by repeatedinjections of PLP in solution is not feasible. Elmer et al. proposedprovision of PLP in a prolonged way over time via an implant consistingof spermaceti and groundnut oil injected via an intramuscular route atthe hip. Nevertheless, this solution was not found to be convincing, itdoes not manage to re-establish the activity actually measured in plasmabeyond 25% of the potential activity and it does not improve in astatistically significant way the anti-tumoral effect of TPL in miceimplanted with a melanoma B-16 tumor. Similar observations were madewith methioninase. Sun et al. (Cancer Research 63, 8377-8383, 2003)ascertain that in vitro, the holoenzyme PLP-rMETase is relatively stablebut that in vivo, this complex is rapidly dissociated leading to a lossof activity of the rMETase. The authors further show that the methioninedepletion duration obtained with rMETase as well as with PEG-rMETase maybe improved by a PLP supplement via the implantation of a PLP pump (apump continuously administering PLP). Nevertheless, this continuousadministration device will invariably be confronted with the problem ofthe low bioavailability of PLP in plasma.

Therefore, although the therapeutic potential of the PLP enzymes hasbeen the subject of much research work, in particular having led formethioninase to conducting pilot clinical trials, no demonstration ofthe clinical efficiency of these enzymes was able to be provided.

Thus, with the purpose of utilizing the therapeutic potential of PLPenzymes, it would be advantageous to have a solution allowing theseenzymes to be maintained in the presence of an optimum and availableamount of PLP.

Various methods have been described for allowing incorporation of activeingredients in erythrocytes. Among these methods, the so-calledlysis-resealing technique is the most widespread. This techniquecomprises three alternatives, which are hypotonic dialysis, hypotonic«preswelling» and hypotonic dilution, all based on the difference inosmotic pressure between the inside and the outside of the erythrocytes.These alternatives have in common the five following steps: packed RedBlood Cells are washed and centrifuged one or several times with aphysiological buffer, the erythrocytes are put into contact with ahypotonic liquid medium resulting in the opening of pores in theerythrocyte membrane, the active ingredient enters the erythrocytes, thepores are closed («resealed») by means of a hypertonic buffer, confiningthe active ingredient inside the erythrocytes, and the latter are thensuspended in a preservation solution. Hypotonic dialysis technique isthe most interesting technique and has been the subject of industrialdevelopments. The one described in EP 1 773 452 is the most performingat the present time, it has the advantage of being reproducible and ofimproving the encapsulation rate of the active ingredient.

The encapsulation of enzymes in erythrocytes, with view to limiting therisks related to immunogenicity of the enzyme, to extending itshalf-life, was already proposed in research work which was the subjectof scientific publications. Encapsulation of an enzyme, L-asparaginase,was described in EP 1 773 452, as well as arginine deiminase in EP 1 874341.

The previous studies do not relate to an enzyme requiring a cofactor anddo not tackle the complexity related to the kinetics of a PLP enzyme andof its PLP cofactor.

An objective of the invention is to provide a pharmaceutical compositioncontaining a PLP enzyme, which allows limitation of the risks related tothe immunogenicity of the enzyme, extension of its half-life, whileputting the enzyme in the presence of an optimal and available amount ofits PLP cofactor.

The object of the invention is thus a suspension of erythrocytes in apharmaceutically acceptable vehicle or a pharmaceutical compositioncomprising erythrocytes and a pharmaceutically acceptable vehicle, theerythrocytes encapsulating a PLP enzyme. This will be hereafter referredto as a composition in order to equally refer to the suspension and thepharmaceutical composition. By «encapsulating» is meant that the activeingredient (enzyme and optionally cofactor and/or other molecule) isessentially or totally present inside. «Essentially» means that aminority proportion of the active ingredient may nevertheless be foundtrapped in the membrane.

The composition notably contains from 0.01 to 30, preferably from 0.05to 10 mg of PLP enzyme per ml of red blood cells.

According to a first embodiment, the PLP enzyme is methioninase, furthercalled, inter alia, L-methioninase, Methionine Gamma Lyase MGL, numberEC 4.4.1.11, CAS number 42616-25-1. In order to be aware of themethioninase sources which may be used according to the invention,mention may notably be made to the publication El Sayed A, AppliedMicrobiol. Biotechnol. (2010) 86: 445-467.

According to a second embodiment, the PLP enzyme is TyrosinePhenol-Lyase or TPL, EC 4.1.99.2, CAS 9059-31-8. Reference may be madeto H. Kumagai et al., J. Biol. Chem. 245, 7: 1767-72 and 245, 7: 1773-7.

According to a third embodiment, the PLP enzyme is tyrosineaminotransferase (hTATase), EC 2.6.1.5, CAS 9014-55-5. Reference may bemade to R. Rettenmeier et al., Nucleic Acids Res. 1990, 18, 13: 3583-61.

According to a fourth embodiment, the PLP enzyme is cystathioninebeta-synthethase or synthase, EC 4.2.1.22, CAS 9023-99-8. Reference maybe made to J. Kraus et al., J. Biol. Chem. 1978, 253, 18: 6523-8.

The composition may further comprise the cofactor of the enzyme, i.e.PLP, and/or a precursor thereof, which may be a non-phosphate precursor,such as a non-phosphate form of vitamin B6, and/or a phosphate precursorsuch as pyridoxine phosphate (PNP).

Vitamin B6 exists in different forms, either phosphate or non-phosphate.Pyridoxine phosphate (PNP), pyridoxal phosphate (PLP) and pyridoxaminephosphate (PMP) are the phosphate forms thereof. The correspondingnon-phosphate forms are pyridoxine (PN), pyridoxal (PL), andpyridoxamine (PM). The non-phosphate forms of vitamin B6 may cross theerythrocyte membrane, which the phosphate forms can only cross withdifficulty. According to the predominant route (as described by Andersonet al. J. Clin. Invest. 1971, Vol. 50, 1901-1909), pyridoxine (PN) istransformed inside the erythrocytes into PNP under the effect ofPN-kinase, PNP is then transformed into PLP under the effect ofPNP-oxidase. The PLP may then be transformed into pyridoxal (PL) underthe effect of PLP-phosphatase and the PL may leave the erythrocytes. Itis easily understood that the provided precursor is able to undergotransformations in the erythrocytes during the preparation method orduring the storage of the composition.

By a non-phosphate form of vitamin B6, will be meant here one of thethree “vitamers” of vitamin B6 or a mixture of two or three vitamers:PL, PN and PM. The PN form is preferred. They may also be in the form ofa salt.

The composition comprises PLP encapsulated in erythrocytes. The PLP maybe provided during the encapsulation procedure or be totally or partlyobtained in the erythrocytes from its precursor. The PLP either presentor formed may be associated with the enzyme. The composition maytherefore comprise the corresponding holoenzyme, for examplemethioninase-PLP. Under these conditions, the half-life of the activeenzyme, as observed for example with the duration of the plasmadepletion of its substrate, is considerably increased. The compositionaccording to the invention notably gives the possibility of preservingenzymatic activity beyond 24 hours after administration, notably at orbeyond 1, 5, 10 or 15 days. By enzymatic activity is notably meant adepletion of more than 20, 30, 40 or 50% of the substrate in the plasma.

In an embodiment, the composition therefore comprises pyridoxalphosphate (PLP) and/or a non-phosphate form of vitamin B6 and/or aphosphate precursor, pyridoxine phosphate (PNP) and/or pyridoxaminephosphate (PMP).

According to a feature, PNP and/or PMP is encapsulated inside theerythrocytes within the composition. This precursor may beco-encapsulated with the enzyme or be totally or partly obtained in theerythrocytes from its own precursor.

The composition notably comprises from about 0.05 to about 600, notablyfrom about 0.5 to about 100, preferably from about 5 to about 50 μmolesof PLP and/or PNP and/or PMP, encapsulated per liter (L) of red bloodcells.

According to a feature, the composition comprises erythrocytesencapsulating the PLP enzyme and PLP and further a non-phosphate PLPprecursor, encapsulated in the erythrocytes, present inside theerythrocytes or present inside and outside the erythrocytes. Thisnon-phosphate precursor may be PN, PL or PM, preferably PN, or a mixtureof two or three of these compounds. The non-phosphate precursor may bepresent inside and/or outside the erythrocytes. The presence of thisnon-phosphate precursor gives the possibility of reaching a remarkablyhigher intra-erythrocyte PLP level than in the absence of thisnon-phosphate precursor.

In an embodiment, the composition comprises erythrocytes encapsulatingthe PLP enzyme and in addition PLP and one of its phosphate precursors,PNP, PLP and/or PMP. This same composition may further compriseadvantageously a non-phosphate precursor, notably PN, as this has justbeen described.

The compositions according to the invention preferably have a hematocritgreater than or equal to 35%, 40% or 45%.

According to an embodiment, the composition comprises erythrocytes and apharmaceutically acceptable vehicle, the erythrocytes encapsulating thePLP enzyme, e.g. methioninase, on the one hand, and, vitamin B6 in anon-phosphate form, preferably PN, on the other hand, for simultaneous,separate or sequential administration. The composition may notably be inthe form of a kit, comprising separately the erythrocytes (suspension)and the vitamin B6 in a non-phosphate form, preferably PN (solution).According to an embodiment, the pharmaceutically acceptable vehicle is a«preservation solution» for erythrocytes, i.e. a solution in which theerythrocytes encapsulating an active ingredient are suspended in theirsuitable form for being stored while awaiting their injection. Apreservation solution preferably comprises at least one agent promotingpreservation of the erythrocytes, notably selected from glucose,dextrose, adenine and mannitol. Advantageously, the preservationsolution contains inorganic phosphate allowing inhibition of theintra-erythrocyte PLP-phosphatase enzyme.

The preservation solution may be an aqueous solution comprising NaCl,adenine and at least one compound from among glucose, dextrose andmannitol. According to a feature, it further comprises an inorganicphosphate.

The preservation solution may comprise NaCl, adenine and dextrose,preferably an AS3 medium. According to a feature, it further comprisesan inorganic phosphate.

The preservation solution may comprise NaCl, adenine, glucose andmannitol, preferably a SAG-Mannitol or ADsol medium. According to afeature, it further comprises an inorganic phosphate.

In particular, the composition or suspension, in a preservationsolution, is characterized by an extracellular hemoglobin levelmaintained at a level equal to or less than 0.5, in particular 0.3,notably 0.2, preferably 0.15, even better 0.1 g/dl at 72 h andpreservation at a temperature comprised between 2 and 8° C.

In particular, the composition or suspension, in a preservationsolution, is characterized by an extracellular hemoglobin levelmaintained at a level equal to or less than 0.5, in particular 0.3,notably 0.2, preferably 0.15, even better 0.1 g/dl for a periodcomprised between 24 h and 20 days, notably between 24 and 72 h andpreservation at a temperature comprised between 2 and 8° C.

The extracellular hemoglobin level is advantageously measured by themanual reference method described in G. B. Blakney and A. J. Dinwoodie,Clin. Biochem. 8, 96-102, 1975. Automatic devices also exist whichallows this measurement to be made with a sensitivity which is specificto them.

In particular, the composition or suspension, in a preservationsolution, is characterized by a hemolysis rate maintained at equal to orless than 2, notably 1.5, preferably 1% at 72 h and preservation at atemperature comprised between 2 and 8° C.

In particular, the composition or suspension, in a preservationsolution, is characterized by a hemolysis rate maintained at equal to orless than 2, notably 1.5, preferably 1% for a period comprised between24 h and 20 days, notably between 24 and 72 h and at a temperaturecomprised between 2 and 8° C.

In particular, the hematocrit of the suspension is equal to or greaterthan 35%, 40%, 45%.

According to a particular method, the metabolism of vitamin B6 inerythrocytes is modified so as to increase the intra-erythrocyte PLPconcentration by increasing the intra-erythrocyte levels of PN-kinaseand PNP-oxidase and/or by reducing the intra-erythrocyte level ofPLP-phosphatase.

According to a characteristic, the composition comprises, in addition tothe PLP enzyme, e.g. methioninase, and to PLP or a precursor thereof,PN-kinase and/or PNP-oxidase and/or an agent inhibiting PLP-phosphatase.These enzymes or agents may be found encapsulated in the erythrocytes orbe found outside and inside the erythrocytes.

This (these) enzymes or agents may also be administered separately, bynotably being mixed with the formulation of non-phosphate vitamin B6when it is separated from the suspension of erythrocytes.

The object of the invention is thus such compositions for use as a drug.

The object of the invention is notably a drug giving the possibility ofproviding to a patient in need thereof, a PLP enzyme and its cofactor,under conditions of good bioavailability, which means that the enzymeand its cofactor are available for each other and in an effective amountso that the enzyme is active and efficient in a therapeutic application.The drug notably aims at depleting or reducing the plasma or circulatingconcentration and/or the concentration at an organ, of a substrate ofthe enzyme.

According to a first sub-object, the drug comprises methioninase andallows depletion or reduction of the plasmatic or circulating methioninein a patient in need thereof. The drug is an anticancer drug, it allowstreatment of a cancer, notably a cancer comprising tumoral cellsauxotrophic for methionine, notably breast, lung, colon, kidney,bladder, melanoma and glioblastoma cancers.

According to a second sub-object, the drug comprises methioninase andallows depletion or reduction of the plasmatic or circulating or hepatichomocysteine in a patient in need thereof. The drug allows treatment ofhomocysteinuria and/or hyperhomocysteinemia and/or associatedpathologies, such as a cardiovascular disease, of the central nervoussystem, of the ocular system and/or of the skeleton (EI-Sayed andShindia Targets in gene therapy Prof. Yongping You ed., 2011).

According to third sub-object of the invention, the drug contains TPLand allows depletion or reduction of the plasmatic or circulatingtyrosine in a patient in need thereof. The drug is an anticancer drug,it allows treatment of a cancer, notably a cancer comprising tumoralcells auxotrophic for tyrosine, notably melanoma.

According to a fourth sub-object of the invention, the drug containshTATase and allows depletion or reduction of the plasmatic orcirculating and/or hepatic tyrosine in a patient in need thereof. Thedrug allows treatment of a rare disease related to a deficiency in thisPLP enzyme, notably the Richner-Hanhart syndrome (tyrosinemia of typeII).

According to a fifth sub-object of the invention, the drug containscystathionine beta-synthase and allows depletion or reduction of theplasmatic or circulating and/or hepatic homocysteine in a patient inneed thereof. The drug allows treatment of homocysteinuria and/orhyperhomocysteinemia and/or associated pathologies, such as acardiovascular disease, a disease of the central nervous system, adisease of the ocular system and/or a disease of the skeleton.

The invention also relates to a method for preparing a pharmaceuticalcomposition comprising erythrocytes encapsulating a PLP enzyme, e.g.methioninase, a pharmaceutically acceptable vehicle, and pyridoxalphosphate (PLP) and optionally a phosphate or non-phosphate PLPprecursor, a method comprising the following steps: optionally, andpreferably, a pellet of red blood cells is washed and centrifuged one orseveral times with a physiological buffer; the erythrocyte suspension isput into contact with a hypotonic liquid medium resulting in the openingof pores in the erythrocyte membrane; the erythrocyte suspension is thenput into contact with the PLP enzyme, e.g. methioninase, before andafter opening the pores; the PLP enzyme, e.g. methioninase, enters theerythrocytes; the pores are closed by means of an isotonic orhypertonic, advantageously hypertonic buffer, and a suspension ofresealed erythrocytes containing the PLP enzyme, e.g. methioninase, iscollected; optionally the erythrocyte suspension is incubated forremoving the most fragile erythrocytes; the erythrocyte suspension iswashed and conditioned with a preservation solution; a method wherein:

-   -   PLP and/or, if present, a PLP phosphate precursor, is        co-encapsulated with the PLP enzyme, e.g. methioninase,    -   if present, the non-phosphate PLP precursor is added to the        suspension of erythrocytes before and/or after opening the        pores, and/or    -   if present, the non-phosphate PLP precursor is added during        incubation or to the preservation solution.

Preferably, some PLP is co-encapsulated with the PLP enzyme and at leastone non-phosphate precursor, such as PN, PL and/or PM, is added to theerythrocyte suspension before and/or after opening the pores, and/orduring incubation and/or to the preservation solution. Preferably, thenon-phosphate precursor is PN.

The erythrocyte suspension is put into contact with a hypotonic liquidmedium resulting in the opening of pores in the erythrocyte membrane. Itis seen that there exist three alternatives in the lysis-resealingtechnique, which are hypotonic dialysis, hypotonic preswelling andhypotonic dilution, all based on the difference in osmotic pressurebetween the inside and the outside of the erythrocytes. Hypotonicdialysis is preferred.

The suspension of erythrocytes encapsulating the PLP enzyme, e.g.methioninase, and optionally PLP and/or a PLP precursor, is notably ableto be obtained with the following method:

-   1—suspending a pellet of erythrocytes in an isotonic solution at a    hematocrit level equal to or greater than 65%, cooling between +1    and +8° C.,-   2—a lysis procedure, at a temperature maintained between +1 and +8°    C., comprising the passing of the suspension of erythrocytes at a    hematocrit level equal or greater than 65% and of a cooled hypotonic    lysis solution between +1 and +8° C., into a dialysis device, such    as a coil or a dialysis cartridge (the cartridge is preferred);-   3—an encapsulation procedure by adding, preferably gradually, the    active ingredient(s) to be encapsulated (notably in a solution made    up beforehand) into the suspension before or during lysis, at a    temperature maintained between +1 and +8° C.; and-   4—a resealing procedure conducted in the presence of an isotonic or    hypertonic, advantageously hypertonic solution, at a higher    temperature, notably comprised between +30 and +42° C.

In a preferred alternative, inspiration may be drawn from the methoddescribed in WO-A-2006/016247 (EP 1 773 452):

1—suspending a pellet of erythrocytes in an isotonic solution at ahematocrit level equal to or greater than 65%, cooling between +1 and+8° C.,

2—measuring osmotic fragility from a sample of erythrocytes from thissame pellet,

3—a lysis procedure, at a temperature maintained between +1 and +8° C.,comprising the passing of the suspension of erythrocytes at a hematocritlevel equal to or greater than 65% and of a hypotonic lysis solutioncooled between +1 and +8° C., into a dialysis device, such as a coil ora dialysis cartridge (the cartridge is preferred); the lysis parametersbeing adjusted according to the osmotic fragility measured earlier;notably, depending on the measured osmotic fragility, the flow of theerythrocyte suspension passing into the dialysis device is adjusted orthe osmolarity of the lysis solution is adjusted; and

4—a procedure for encapsulation by adding, preferably gradually, theactive ingredient(s) to be encapsulated (notably in a solution madebeforehand) in the suspension before and during lysis, at a temperaturemaintained between +1 and +8° C.; and

5—a resealing procedure conducted in the presence of an isotonic orhypertonic, advantageously hypertonic solution, at a higher temperature,notably comprised between +30 and +42° C.

Notably, for dialysis, the pellet of erythrocytes is suspended in anisotonic solution with a high hematocrit level, equal to or greater than65%, and preferably equal to or greater than 70%, and this suspension iscooled between +1 and +8° C., preferably between +2 and +6° C.,typically around +4° C. According to a particular method, the hematocritlevel is comprised between 65 and 80%, preferably between 70 and 80%.

When it is measured, the osmotic fragility is advantageously measured onerythrocytes just before the lysis step, in the presence or in theabsence, preferably in the presence of the active ingredient(s) to beencapsulated. The erythrocytes or the suspension containing them areadvantageously at a temperature close to, or identical with thetemperature selected for lysis. According to another advantageousfeature of the invention, the conducted measurement of the osmoticfragility is rapidly utilized, i.e. the lysis procedure is carried outin a short time after taking the sample. Preferably, this lapse of timebetween the sampling and beginning of lysis is less than or equal to 30minutes, still better less than or equal to 25 and even to 20 minutes.

As regards to how to conduct the lysis-resealing procedure withmeasurement and taking into account of the osmotic fragility, oneskilled in the art may refer for more details to WO-A-2006/016247. Thisdocument is incorporated herein by reference.

An enhancement of the encapsulation techniques was described in FR 1 354204 filed on May 7, 2013, to which one skilled in the art may refer andwhich is incorporated herein by reference. Thus, according to anembodiment, the erythrocytes encapsulating the active ingredients, i.e.the PLP enzyme, e.g. methioninase, and optionally one or several otheractive ingredients such as PLP and/or a PLP precursor, are obtained by amethod comprising the encapsulation of the active ingredient insideerythrocytes by lysis-resealing, the obtaining of a suspension or of apellet comprising erythrocytes incorporating the active ingredient and asolution with an osmolality greater than or equal to 280 mOsmol/kg, inparticular between about 280 and about 380 mOsmol/kg, preferably betweenabout 290 and about 330 mOsmol/kg, the incubation of the pellet or ofthe suspension as such or after adding an incubation solution, at anosmolality greater than or equal to 280 mOsmol/kg, in particular betweenabout 280 and about 380 mOsmol/kg, preferably between about 290 andabout 330 mOsmol/kg. Incubation is notably carried out for a periodgreater than or equal to 30 minutes, in particular greater than or equalto 1 h. It is then proceeded with removal of the liquid medium of theincubated solution and the erythrocytes obtained are suspended in asolution allowing injection of the suspension into a patient, preferablya preservation solution allowing injection of the suspension into apatient. The indicated osmolality is that of the solution in which theerythrocytes are suspended or in a pellet at the relevant moment.

According to a particular method, a non-phosphate PLP precursor, notablya non-phosphate form of vitamin B6 is provided during the production orstorage method or in the final formulation. This compound may forexample be incorporated into the incubation solution or into thepreservation solution, or further into the formulation before injectionwhen a pre-injection dilution is carried out.

According to a feature, notably 0.1 to 250, preferably from 1 to 50 mMof PN and/or of PL and/or of PM are provided during the production orstorage method or in the final formulation. As described above, afraction of these non-phosphate derivatives of vitamin B6 will beconverted into PLP in the red blood cells.

By «stabilized erythrocyte suspension», is notably meant a suspensionhaving an extracellular hemoglobin content which remains less than orequal to 0.2 g/dl until its use in humans, the latter may intervenenotably from 1 to 72 hours after producing the erythrocyte batchincorporating the active ingredient.

By «ready-to-use stabilized erythrocyte suspension», is meant thestabilized suspension in a solution allowing injection into a patient,notably in a preservation solution. Its hematocrit is generally equal toor greater than 35%, 40% or 45%.

By «erythrocyte pellet is meant a concentrate or concentration oferythrocytes collected after separating the erythrocytes of the liquidmedium in which they were suspended previously. The separation may beensured by filtration or by centrifugation. Centrifugation is the meansgenerally used for such a separation. A pellet comprises a certainproportion of liquid medium. Generally, the pellet has a hematocritcomprised between 70 and 85%.

By «incubation solution», is meant the solution in which theerythrocytes encapsulating an active ingredient are present during theincubation step. The incubation may be accomplished over a large rangeof hematocrits, notably between 10 and 85% of hematocrit.

By «fragile erythrocytes», are meant the erythrocytes stemming from theincorporation procedure which may, once suspended in a preservationsolution, be lyzed when the suspension is preserved between 2 and 8° C.,notably after 1 to 72 h.

By «initial hematocrit is meant the hematocrit before cell loss due tolysis of the fragile erythrocytes during incubation.

The method may notably comprise the following steps:

-   (a) encapsulation of the active ingredient(s) to be encapsulated    (PLP enzyme, e.g. methioninase, and optionally PLP and/or a PLP    precursor) inside erythrocytes, comprising the putting of the    erythrocytes into contact with a hypotonic medium (allowing opening    of pores in the membrane of the erythrocytes), the contacting with    the active ingredient (for allowing it to enter the erythrocytes),    the resealing of the erythrocytes, notably by means of an isotonic    or hypertonic medium, advantageously hypertonic,-   (b) obtaining or preparing a suspension or pellet comprising    erythrocytes incorporating the active ingredient and a solution with    an osmolality greater than or equal to 280 mOsmol/kg, in particular    between about 280 and about 380 mOsmol/kg, preferably between about    290 and about 330 mOsmol/kg,-   (c) incubating the pellet or the suspension of step (b) as such or    after adding an incubation solution, at an osmolality greater than    or equal to 280 mOsmol/kg, in particular between about 280 and about    380 mOsmol/kg, preferably between about 290 and about 330 mOsmol/kg,    for a period greater than or equal to 30 minutes, notably greater    than or equal to 1 h,-   (d) removing the liquid medium of the incubated suspension of step    (c),-   (e) suspending the erythrocytes obtained under (d) into a solution    allowing injection of the suspension into a patient, preferably a    preservation solution allowing injection of the suspension into a    patient.

The vitamin B6 in the non-phosphate form may be added during theencapsulation step, in step (a) or during the incubation in step (c) orfurther in the preservation solution.

According to a first method, the step following the encapsulation bylysis-resealing, notably step (b), includes at least 1 washing cycle,preferably 2 or 3 washing cycles, by dilution of the obtained suspensionor pellet in the lysis-resealing step or step (a) in a solution, at anosmolality greater than equal to 280 mOsmol/kg, in particular betweenabout 280 and about 380 mOsmol/kg, preferably between about 290 andabout 330 mOsmol/kg, and then obtaining a pellet of erythrocytes or asuspension. This pellet or this suspension comprises erythrocytesincorporating the active ingredient and a solution with an osmolalitygreater than or equal to 280 mOsmol/kg, in particular between about 280and about 380 mOsmol/kg, preferably between about 290 and about 330mOsmol/kg. The following steps, e.g. (c), (d) and (e) are then applied.

According to a second method, in the lysis-resealing step or step (a),resealing of the erythrocytes by means of an isotonic or hypertonicmedium produces the suspension of erythrocytes which may then be subjectto incubation, e.g. the suspension of step (b), in a solution with anosmolality greater than or equal to 280 mOsmol/kg, in particular betweenabout 280 and about 380 mOsmol/kg, preferably between about 290 andabout 330 mOsmol/kg. In other words, the lysis-resealing step or step(a) includes a step for resealing the erythrocytes wherein the suspendederythrocytes encapsulating an active ingredient are mixed with anisotonic or hypertonic resealing solution, advantageously hypertonic,producing a suspension of erythrocytes with an osmolality greater thanor equal to 280 mOsmol/kg, in particular between about 280 and about 380mOsmol/kg, preferably between about 290 and about 330 mOsmol/kg. In thismethod, the incubation step or step (c) comprises incubation of thesuspension stemming from the resealing. The incubation is carried outfor a period greater than or equal to 30 minutes, notably greater thanor equal to 1 h. The following steps, e.g. (d) and (e) are then applied.

The steps following the lysis-resealing, e.g. (b) to (e), are conductedunder conditions resulting in the lysis of fragile erythrocytes, or of amajority of them, notably more than 50, 60, 70, 80 or 90%, or more. Todo this, it is possible to act on the incubation period, the incubationtemperature and on the osmolality of the solution in which theerythrocytes are suspended. The higher the osmolality, the longer theincubation time may be. Thus the lower the osmolality, the shorter maybe the incubation in order to obtain the same effect. Also, the higherthe temperature, the shorter the incubation time may be, and vice versa.One or several washing cycles will then allow removal of cell debris andextracellular hemoglobin, as well as the extracellular activeingredient.

According to the invention, a washing cycle comprises the dilution ofthe suspension or pellet of erythrocytes, and then the separationbetween the erythrocytes and the washing solution. Preferably, a washingstep comprises preferably 2 or 3 dilution-separation cycles. Theseparation may be achieved by any suitable means, such as filtration andcentrifugation. Centrifugation is preferred.

Incubation is not limited by the hematocrit of the suspension. In thisway, a suspension having an initial hematocrit generally comprisedbetween 10 and 85%, notably between 40 and 80% may be incubated. This israther referred to as a pellet from 70% and as a suspension below thisvalue.

The removal step or step (d) aims at removing the liquid portion of thesuspension or of the incubated pellet, in order to notably remove celldebris and the extracellular hemoglobin, as well as consequently theextracellular active ingredient.

According to a first method for the removal step or step (d),separation, notably centrifugation is carried out, this being notablyapplicable to a suspension. This separation may be followed by one orseveral, for example 2 or 3, washing cycles, by dilution in an isotonicsolution, and then separation, notably by centrifugation.

According to a second method for the removal step or step (d), dilutionbefore separation notably centrifugation is carried out, this beingapplicable to a suspension or to a pellet. The dilution may notably becarried out with an isotonic washing solution or with a preservationsolution.

The final step or step (e) consists of preparing the final suspensionsuch that it may be administered to the patient, without any othertreatment.

According to a first method for this step, a dilution of the erythrocytepellet from the removal step or step (d) is carried out with theinjection solution, notably the preservation solution.

According to a second method for this step, one or several cycles forwashing the erythrocyte pellet stemming from the removal step or step(d) is carried out with the injection solution, notably the preservationsolution, by dilution followed by separation. After washing, theerythrocytes are re-suspended in the injection solution, notably thepreservation solution.

The method of the invention may further comprise one, several or thetotality of the following features:

-   -   the incubation step or step (c) is carried out at a temperature        comprised between about 2 and about 39° C., over sufficient time        for ensuring lysis of fragile erythrocytes;    -   the incubation step or step (c) is carried out at a low        temperature, notably comprised between about 2 and about 10° C.,        in particular between about 2 and about 8° C., and lasts for        about 1 h to about 72 h, notably from about 6 h to about 48 h,        preferably from about 19 h to about 30 h;    -   the incubation step or step (c) is conducted at a higher        temperature comprised between about 20 and about 39° C., notably        at room temperature (25° C.±5° C.) and lasts for about 30 min to        about 10 h, notably from about 1 h to about 6 h, preferably from        about 2 h to about 4 h; it is possible to operate at an even        higher temperature than room temperature, but this may have a        negative impact on the cell yield, P50 and/or the 2,3-DPG        content;    -   in the incubation step or step (c), the suspension is at an        initial hematocrit comprised between 10 and 85%, notably between        40 and 80%; a pellet from separation, having for example a        hematocrit between 70 and about 85%, or a diluted pellet having        a hematocrit comprised between about 40 and 70% may be        incubated;    -   the incubation step comprises stirring of the suspension;    -   the incubation step does not comprise any stirring;    -   as a solution for washing and/or incubation, a metered aqueous        NaCl solution is used for obtaining the desired osmolality; as        an example, a solution may thus comprise 0.9% of NaCl; this        solution may also comprise, notably in addition to NaCl,        glucose, notably glucose monohydrate, monosodium phosphate        dihydrate, disodium phosphate dodecahydrate; as an example, a        composition comprises: 0.9% of NaCl, 0.2% of glucose        monohydrate, 0.034% of monosodium phosphate dihydrate, 0.2% of        disodium phosphate dodecahydrate;    -   the washing in the final step or step (e) is carried out with        the preservation solution;    -   the osmolality of the solution (liquid portion) in the        ready-to-use suspension or which may be injected into the        patient is comprised between about 280 and about 380 mOsmol/kg,        preferably between about 290 and about 330 mOsmol/kg;    -   the hematocrit of the ready-to-use suspension or which may be        injected into the patient is equal to or greater than 35%, 40%        or 45%;    -   all the steps for washing, incubation are carried out with the        preservation solution;    -   the washing solution of step (b) and/or the washing solution of        step (e) and the preservation solution are of the same        composition and comprise compound(s) promoting preservation of        the erythrocytes;    -   the preservation solution (and the washing solution(s) or the        incubation solutions if necessary) is an aqueous solution        comprising NaCl, adenine and at least one compound from among        glucose, dextrose and mannitol;    -   the preservation solution (and the washing or incubation        solution(s) if necessary) comprises NaCl, adenine and dextrose,        preferably an AS3 medium;    -   the preservation solution (and the washing or incubation        solution(s), if necessary) comprise NaCl, adenine, glucose and        mannitol, preferably a SAG-Mannitol or ADsol medium.

The methods according to the invention notably comprise the followingstep:

-   (a) encapsulating an active ingredient inside erythrocytes,    comprising the contacting with a hypotonic medium allowing opening    of pores in the membrane of the erythrocytes, the contacting with    the active ingredient in order to allow its entry into the    erythrocytes, the resealing of the erythrocytes by means of an    isotonic or hypertonic medium. It should be noted that the active    ingredient may be present in the suspension of erythrocytes before    the lysis of the latter, or further be added during lysis or after    lysis, but always before resealing. In an embodiment of this step    (a), the method comprises the following sub-steps:-   (a1) having a suspension of erythrocytes at a hematocrit equal to or    greater than 60 or 65%,-   (a2) measuring the osmotic fragility of the erythrocytes in this    suspension,-   (a3) a procedure for lysis and internalization of the active    ingredient(s), comprising the passing of the erythrocyte suspension    into a dialysis device, notably a dialysis cartridge, counter to a    lysis solution, adjusting the flow of the erythrocyte suspension or    adjusting the flow rate of the lysis solution or adjusting the    osmolarity of the lysis solution, depending on the osmotic fragility    measured under (a2),-   (a4) a procedure for resealing the erythrocytes.

Another object of the invention is a therapeutic treatment methodintended to provide a patient in need thereof, with a PLP enzyme and itscofactor, under conditions of good bioavailability, which means that theenzyme and its cofactor are available for each other and in an effectiveamount so that the enzyme is active and efficient in a therapeuticapplication. This method notably aims at depleting or reducing theplasmatic or circulating concentration and/or the concentration at anorgan, of a substrate of the enzyme. This method comprises theadministration of an effective amount of a composition according to theinvention or the use of a kit according to the invention.

According to a first sub-object, the invention is a therapeutictreatment method allowing depletion or reduction of plasmatic orcirculating methionine in a patient in need thereof. This methodcomprises the administration of an effective amount of a compositionaccording to the invention or the use of a kit according to theinvention, comprising methioninase and its cofactor. The method is amethod for treating cancer, notably a cancer comprising tumoral cellsauxotrophic for methionine, notably breast, lung, colon, kidney,bladder, melanoma and glioblastoma cancers.

According to a second sub-object, the invention is a therapeutictreatment method allowing depletion or reduction in plasmatic orcirculating or hepatic homocysteine in a patient in need thereof. Thismethod comprises the administration of an effective amount of acomposition according to the invention or the use of a kit according tothe invention, comprising methioninase and its cofactor. The method is amethod for treating homocysteinuria and/or a hyperhomocysteinemia and/orpathologies associated with hyperhomocysteinemia, such as acardiovascular disease, a disease of the central nervous system, adisease of the ocular system and/or a disease of the skeleton.

According to a third sub-object of the invention, the invention is atherapeutic treatment method allowing depletion or reduction in theplasmatic or circulating tyrosine in a patient in need thereof. Thismethod comprises the administration of an effective amount of acomposition according to the invention or the use of a kit according tothe invention, comprising TPL and its cofactor. The method is a methodfor treating a cancer, notably a cancer comprising tumoral cellsauxotrophic for tyrosine, notably melanomas.

According to a fourth sub-object of the invention, the invention is atherapeutic treatment method allowing depletion or reduction of theplasmatic or circulating and/or hepatic tyrosine in a patient in needthereof. This method comprises the administration of an effective amountof a composition according to the invention or the use of a kitaccording to the invention, comprising hTATase and its cofactor. Themethod is a method for treating a rare disease related to a deficiencyof this PLP enzyme, notably the Richner-Hanhart syndrome (tyrosinemia oftype II).

According to a fifth sub-object of the invention, the invention is atherapeutic treatment method allowing depletion or reduction inplasmatic or circulating and/or hepatic homocysteine in a patient inneed thereof. This method comprises the administration of an effectiveamount of a composition according to the invention or the use of a kitaccording to the invention, comprising cystathionine beta-synthase andits cofactor. The method is a method for treating homocysteinuria and/orhyperhomocysteinemia and/or pathologies associated withhyperhomocysteinemia, such as a cardiovascular disease, a disease of thecentral nervous system, a disease of the ocular system and/or a diseaseof the skeleton.

The composition used in these therapeutic applications may furthercomprise the cofactor of this PLP enzyme, i.e. PLP, and/or a precursorthereof, which may be a non-phosphate precursor, such as a non-phosphateform of vitamin B6, and/or a phosphate precursor, such as pyridoxinephosphate (PNP). The composition may also comprise PN-kinase,PNP-oxidase, an agent inhibiting PLP-phosphatase. More generally thetreatment method may comprise the administration of a composition or akit as described above.

One administer to the patient per month of treatment, one or severaldoses, notably one or two, representing 50 to 300 ml of suspension orcomposition with a hematocrit greater than or equal to 35%, 40% or 45%,in one or several injections. They are notably administered byintravenous or intra-arterial injection, notably by perfusion.

Alternatively, an effective amount of a composition comprisingerythrocytes encapsulating the PLP enzyme, e.g. methioninase, and aneffective amount of a solution containing a non-phosphate form ofvitamin B6, preferably PN are administered separately to the samepatient. This non-phosphate form of vitamin B6 may be administered byinjection, either simultaneously or separately with the suspension oferythrocytes or via any other route, notably an oral route.

In a first embodiment, a suspension of erythrocytes encapsulating theactive ingredient(s), prepared within 1 and 72 h, notably for between 10and 72 h before injection is injected to the patient. This suspensionhas a hematocrit equal to or greater than 35%, 40% or 45%. It is foundin a preservation solution. The extracellular hemoglobin level is equalto or less than 0.5, in particular 0.3, notably 0.2, preferably 0.15,still better 0.1 g/dl, and/or the hemolysis level equal to or less than2, notably 1.5, preferably 1%. The suspension is not washed or subjectto a similar operation before injection.

Another object of the invention is a process for producing methioninase,under purified form, and with high yield, comprising the steps of:

-   (a) culturing bacteria transformed to produce methioninase,    centrifugation of the culture and recovering of the pellet,-   (b) suspending the pellet in a lysis buffer and lysis of the    bacteria cells, centrifugation and recovery of the supernatant,-   (c) treating the supernatant with a precipitating agent,    precipitation and recovery of the pellet,-   (d) applying to the pellet two rounds of crystallization or    precipitation using PEG at a temperature comprised between about 25    and 40° C., recovering the pellet,-   (e) suspending the pellet in a solubilization buffer (for example    [25 mM Tris; 0.5 mM P5P; 0.5 mg/mL beta mercapto ethanol; pH 7.5])    and subjecting to two rounds of anion exchange chromatography,    recovering a solution of methioninase,-   (f) submitting the solution of methioninase to a polishing step by    chromatography, recovery of a purified methioninase solution.

In a preferred embodiment, use is made of the methioninase codingsequence of Pseudomas putida. This sequence may be optimized in order toadapt the sequence to the production strain. The production strain ispreferably E. coli, such as strain HMS174. An expression vectorcontaining the methioninase sequence, preferably the optimized one, isused to transform the producing strain, and a producing clone may beselected. Then production of methioninase using this clone is performedin a fermenter under usual conditions.

Preferably, the pellet of step (a) is resuspended in the lysis buffer(for example [100 mM Sodium phosphate; 4.4 mM EDTA; 3.3 mM P5P; 1 mMDTT; pH 7.6]) (7 mL per gram of wet weight). Preferably, lysis is madeby high pressure homogenization, advantageously in several steps,preferably 3 steps of high pressure homogenization. Typical temperatureis maintained about 10° C. before each step of homogenization (between 9to 12° C.). Preferably, after lysis and before centrifugation, the celllysate is submitted to clarifying using a cationic coagulant, preferablypolyethyleneimine (PEI). Typical PEI concentration may be between about0.05 and about 0.5% (VN), in particular between about 0.1 and about 0.3%preferably about 0.2%.

Precipitation at step (c) may be performed with ammonium sulfate,typically at about 60% saturation. Preferably, before thisprecipitation, the supernatant is filtered on an about 0.2 μm membrane.

At step (d), PEG is preferably PEG-6000. Its final concentration may bebetween about 5 and about 25% (WN), in particular between about 5 andabout 15%. The first round may preferably be performed in the presenceof ammonium sulfate. Typically, ammonium sulfate may be at about 10%saturation (between 9 to 11%). Typically, PEG may be at about 10% finalconcentration. The second round may preferably be performed in thepresence of an inorganic salt, typically an alkaline metal salt such assodium chloride or potassium chloride, preferably sodium chloride. Thesalt may be at a final concentration of about 0.20 M (between 0.19 and0.21). Typically, PEG may be at about 12% final concentration.Temperature may be comprised between about 25 and about 35° C., inparticular between about 28 and about 32° C., typically about 30° C.

At step (e) chromatography may be performed on DEAE sepharose.Preferably, before chromatography, the resuspended pellet or sedimentmay be submitted to passage through an about 0.45 μm filter.

At step (f), polishing is performed to remove remaining residualcontaminants such as endotoxins, HCP and DNA. It may be performed usinga Q membrane chromatography.

The purified methioninase may then be concentrated and diafiltered.Conservation may be made through freeze-drying and storage at about −80°C. The invention will now be described in more detail by means ofembodiments taken as non-limiting examples and with reference to thedrawing wherein:

FIG. 1: Description of the method for purifying MGL, according to themethod described in EP 0 978 560 and according to the improved methoddescribed in the present application. The modifications brought to themethod described in patent EP 0 978 560 B1 relate to the steps locatedafter the precipitation step with ammonium sulfate.

FIGS. 2 and 3. Comparison of the intra-erythrocyte concentrations (FIG.2) and extracellular concentrations (FIG. 3) of PLP after incubation ofa RC-MGL-PLP suspension with pyridoxine (PN) at differentconcentrations. The RC-MGL-PLP suspension, incubated for 3 h and 24 h atroom temperature in the absence of pyridoxine (0 mM) has a basal PLPlevel of about 3.9 μM. The incubation of the suspensions with 2 mM and 4mM pyridoxine gives the possibility of increasing the intra-erythrocytePLP concentration to 8 μM after 3 h of incubation (pale grey bars) andgives the possibility of attaining considerably higher levels (11 μM and14 μM respectively) after 24 h of incubation (dark grey bars).

FIG. 4. Pharmacokinetics of red corpuscles (RCs) loaded with a complexMGL-PLP. The RC-MGL-PLP2 product is obtained by lysis-resealing of asuspension containing 3 mg/ml of MGL and ˜30 μM of PLP. The RC-MGL-PLP3product is obtained by lysis-resealing of a suspension containing 3mg/ml of MGL and ˜125 μM of PLP. The RC-MGL-PLP4 product is obtained bylysis-resealing of a suspension containing 5 mg/ml of MGL and 33 μM ofPLP. The RC-MGL-PLP5 product is obtained by lysis-resealing of asuspension containing 6 mg/ml of MGL and 100 μM of PLP. Fluorescentlabeling of the products (CFSE) allows traceability of the RCs in vivo.The products injected intravenously to the mice CD1 (8 ml/kg for theproducts RC-MGL-PLP2, RC-MGL-PLP3 and RC-MGL-PLP5 and 10 ml/kg for theproduct RC-MGL-PLP4) have excellent stability with a survival rate ofthe injected RCs greater than 75% at 120 h, i.e. 5 days after theiradministration. For the RC-MGL-PLP4 product, the survival rate isreduced to less than 75% after ˜10 days.

FIG. 5. Pharmacodynamics of the free MGL enzyme. The MGL enzyme wasdiluted by means of a potassium phosphate solution supplemented with 10μM of P5P in order to obtain two injectable products (MGL-L1 andMGL-L2). These products were made so as to obtain 1) the same enzymeconcentration as the product RC-MGL-PLP2, i.e. 0.45 mg/ml of MGL and 2)a concentration twice greater than the product RC-MGL-PLP2, i.e. 0.90mg/ml of MGL. Both products are administered intravenously (IV) to CD1mice (8 ml/kg) with supplementation IV of pyridoxine 6 h after theinjection. The plasma L-methionine level is measured by HPLC-MS-MS. TheL-Met level in non-treated CD1 was evaluated to be 68 μM. These productsMGL-L1 and MGL-L2 both lead to rapid depletion within 15 min after theiradministration but not long lasting over time.

FIG. 6. Pharmacodynamics of the RC-MGL-PLPs over short times. Theproduct RC-MGL-PLP2 is obtained by lysis-resealing of a suspensioncontaining 3 mg/ml of MGL and ˜30 μM of PLP. The product RC-MGL-PLP3 isobtained by lysis-resealing of a suspension containing 3 mg/ml of MGLand ˜125 μM of PLP. Both products are administered intravenously (IV) toCD1 mice (8 ml/kg) with IV supplementation of pyridoxine 6 h afteradministration for the mice receiving RC-MGL-PLP2. The plasmaL-methionine level is measured by HPLC-MS-MS. The L-Met level inuntreated CD1s was evaluated to be 82 μM. Both products RC-MGL-PLP2 andRC-MGL-PLP3 lead to rapid depletion 15 min after their administrationreducing the L-Met level to 15.0±3.6 μM and 22.7±1.5 μM respectively andthen maintaining more moderate depletion at 35 μM but stable between 48h and 120 h.

FIG. 7. Pharmacodynamics of the RC-MGL-PLPs over long periods. Theproduct RC-MGL-PLP4 (0.5 mg/ml) is obtained by lysis-resealing of asuspension containing 5 mg/ml of MGL and 33 μM of PLP. The product isadministered intravenously (IV) to CD1 mice (10 ml/kg) with IVsupplementation of pyridoxine 6 h after administration for the micereceiving RC-MGL-PLP4. The plasma L-methionine level is measured byHPLC-MS-MS. The L-Met level in untreated CD1s was evaluated to be 68 μM.The product RC-MGL-PLP4 leads to rapid depletion 15 min afteradministration reducing the L-Met level to ˜10 μM and then maintainingmore moderate depletion at ˜25 μM but stable between 24 h and 48 h so asto then gradually return to the control values 12 days after injection.

FIG. 8. Residual activity of circulating PLP enzymes. The enzyme MGL wasdiluted by means of a potassium phosphate solution supplemented with 10μM of P5P in order to obtain the injectable product MGL-L2. The productRC-MGL-PLP2 is obtained by lysis-resealing of a suspension containing 3mg/ml of MGL and ˜30 μM of PLP. The product RC-MGL-PLP3 is obtained bylysis-resealing of a suspension containing 3 mg/ml of MGL and ˜125 μM ofPLP. The product RC-MGL-PLP4 is obtained by lysis-resealing of asuspension containing 5 mg/ml of MGL and 33 μM of PLP. The productRC-MGL-PLP5 is obtained by lysis-resealing of a suspension containing 6mg/ml of MGL and 100 μM of PLP. The products are injected intravenouslyto CD1 mice (8 ml/kg for the products MGL-L2, RC-MGL-PLP2, RC-MGL-PLP3and RC-MGL-PLP5 and 10 ml/kg for the product RC-MGL-PLP4). The residualactivity of the injected MGL enzyme (total RCs) is determined by ameasurement of the NH₃ produced by MGL according to the method describedin Example 4.

EXAMPLE 1. METHOD FOR OBTAINING AND CHARACTERIZING METHIONINE GAMMALYASE (MGL)

Production of the strain and isolation of a hyper-producing clone: thenatural sequence of MGL of Pseudomonas putida (GenBank: D88554.1) wasoptimized by modifying rare codons (in order to adapt the sequencestemming from P. putida to the production strain Escherichia coli).Other changes have been made to improve the context of translationinitiation. Finally, silent mutations were performed to remove threeelements that are part of a putative bacterial promoter in the codingsequence (box-35, box-10 and a binding site of a transcription factor inposition 56). The production strain E. coli HMS174 (DE3) was transformedwith the expression vector pGTPc502_MGL (promoter T7) containing theoptimized sequence and a producing clone was selected. The producingclone is pre-cultivated in a GY medium+0.5% glucose+kanamycin for 6-8 h(pre-culture 1) and 16 h (pre-culture 2) at 37° C.

Fermentation: the production is then achieved in a fermenter with GYmedium, with stirring, controlled pressure and pH from the pre-culture 2at an optical density of 0.02. The growth phase (at 37° C.) takes placeuntil an optical density of 10 is obtained and the expression inductionis achieved at 28° C. by adding 1 mM IPTG into the culture medium. thecell sediment is harvested 20 h after induction in two phases: the cellbroth is concentrated 5-10 times after passing over a 500 kDa hollowfiber and then cell pellet is recovered by centrifugation at 15900×g andthen stored at −20° C.

Purification: The cell pellet is thawed and suspended in lysis buffer(7v/w). Lysis is performed at 10° C. in three steps by high pressurehomogenization (one step at 1000 bars, and then two steps at 600 bars).The cell lysate then undergoes clarification at 10° C. by adding 0.2%PEI and centrifugation at 15900×g. The soluble fraction is thensterilized by 0.2 μm before precipitation with ammonium sulfate (60%saturation) at 6° C., over 20 h. Two crystallization steps are carriedout on the re-solubilized sediment using solubilization buffer, thefirst crystallization step is realized by addition of PEG-6000 at 10%(final concentration) and ammonium sulfate at 10% saturation, and thesecond crystallization is then performed by addition of PEG-6000 at 12%final concentration and 0.2M NaCl (final concentration) at 30° C. Thepellets containing the MGL protein are harvested at each stage aftercentrifugation at 15900×g. The pellet containing the MGL protein isre-suspended in a solubilization buffer and passed over a 0.45 μm filterbefore being subject to two anion exchange chromatographies (DEAEsepharose FF). The purified protein is then subject to a polishing stepand passed over a Q membrane chromatography capsule for removing thedifferent contaminants (endotoxins, HCP host cell protein, residualDNA). Finally, the purified MGL protein is concentrated at 40 mg/ml anddiafiltered in formulation buffer using a 10 kDa cut-off tangential flowfiltration cassette. Substance is then aliquoted at ˜50 mg of proteinper vial, eventually freeze-dried under controlled pressure andtemperature, and stored at −80° C.

Characterization: The specific activity of the enzyme is determined bymeasuring the produced NH₃ as described in example 4. The purity isdetermined by SDS-PAGE. The PLP level after being taken up in water wasevaluated according to the method described in example 5. The osmolarityis measured with an osmometer (Micro-Osmometer Loser Type 15).

The following table summarizes the main characteristics of one producedbatch of MGL:

MGL of P. putida Formulation Freeze-dried (amount per tube: 49.2 mg).Characteristics after being taken up in 625 μL of water: 78.7 mg/ml,~622 μM of PLP, 50 mM of Na phosphate, pH 7.2, Osmolarity 300 mOsmol/kg.Specific activity 13.2 IU/mg Purity >98%

Discussion of the production method. The method for purifying the MGLdescribed in Example 1 is established on the basis of the methoddetailed in patent EP 0 978 560 B1 and of the associated publication(Takakura et al., Appl Microbiol Biotechnol 2006). This selection isexplained by the simplicity and the robustness of the crystallizationstep which is described as being particularly practical and easilyadaptable to large scale productions according to the authors. This stepis based on the use of PEG6000 and of ammonium sulfate after heating theMGL solution obtained after the lysis/clarification and removal ofimpurities by adding PEG6000/ammonium sulfate steps. The other notablepoint of this step is the possibility of rapidly obtaining a high puritylevel during the step for removing the impurities by achievingcentrifugation following the treatment of the MGL solution with PEG6000.The impurities are again found in the centrifugation pellet, the MGLbeing in majority found in solution in the supernatant. Because of thispurity, the passing of the MGL solution in a single chromatography stepover an anion exchanger column (DEAE), associated with a purificationstep by gel filtration on a sephacryl S200 HR column, gives thepossibility of obtaining a purified protein.

Upon setting into place the patented method for small scale tests, itappeared that the obtained results were not able to be reproduced.According to patent EP 0 978 560 B1, at the end of the step for removingthe impurities (treatment with PEG6000/ammonium sulfate andcentrifugation), the MGL enzyme is in majority found in the solublefraction, centrifugation causing removal of the impurities in thepellet. During small scale tests conducted according to the describedmethod in EP 0 978 560 B1, the MGL protein is again in majority found(˜80%) in the centrifugation pellet. The table below lists thepercentage of MGL evaluated by densitometry on SDS-PAGE gel in solublefractions.

MGL percentage in Purification the soluble fraction Average Test no. 111% 17% Test no. 2 23%

This unexpected result therefore led to optimization of the patentedmethod by: 1) operating from the centrifugation pellet containing MGL,2) carrying out two successive crystallization steps for improving theremoval of the impurities after loading on a DEAE column, 3) optimizingchromatography on a DEAE column.

For this last step, it is found that the DEAE sepharose FF resin isfinally not a sufficiently strong exchanger in the tested buffer and pHconditions. After different additional optimization tests, the selectionwas finally directed to 1) replacement of the phosphate buffer used inthe initial method with Tris buffer pH 7.6 for improving the robustnessof the method and 2) carrying out a second passage on DEAE in order tosubstantially improve the endotoxin level and the protein purity withoutany loss of MGL (0.8 EU/mg according to Takakura et al., 2006 versus0.57 EU/mg for the modified method).

Finally, in order to obtain a method compatible with the requirementsfor large scale GMP production, a polishing step on a membrane Q wasadded in order to reduce the residual endotoxins and HCP levels. Thisfinal step of polishing avoids the implementation of the S200 gelfiltration chromatography which is a difficult step to be used inproduction processes at an industrial scale (cost and duration of thechromatography).

The different purification steps of the method from EP 0 978 560 B1 aswell as of the method of the present application are given in FIG. 1.

The following table gives the possibility of checking that the providedadaptations have led to obtaining a purification method with a yield atleast equivalent to the one described in the initial method.

Patent EP 978 560 B1 Method of the application Amount of Yield Amount ofYield Step enzyme (g) (%) enzyme (g) (%) Solubilised pellet 125 100 70100 before DEAE Concentrated solution^($) 80 64 46 65 ^($)post sephacrylS-200 HR (EP 978 560) or post Membrane Q (method of the invention).

EXAMPLE 2. CO-ENCAPSULATION OF MGL AND PLP IN MURINE ERYTHROCYTES

Whole blood of CD1 mice (Charles River) is centrifuged at 1000×g, for 10min, at 4° C. in order to remove the plasma and buffy coat. The RCs arewashed three times with 0.9% NaCl (v:v). The freeze-dried MGL isre-suspended in water at a concentration of 78.7 mg/ml and added to theerythrocyte suspension in order to obtain a final suspension with ahematocrit of 70%, containing different concentrations of MGL and of thePLP. The suspension was then loaded on a hemodialyzer at a flow rate of120 ml/h and dialyzed against a hypotonic solution at a flow rate of 15ml/min as a counter-current. The suspension was then resealed with ahypertonic solution and then incubated for 30 min at 37° C. After threewashes in 0.9% NaCl, 0.2% glucose, the suspension was taken up in apreservation solution SAG-Mannitol supplemented with 6% BSA. Theobtained products are characterized at D0 (within the 2 h followingtheir preparation) and at D1 (i.e. after ˜18 h-24 h of preservation at2-8° C.). The hematologic characteristics are obtained with a veterinaryautomaton (Sysmex, PocH-100iV).

Results:

In the different studies mentioned hereafter, the MGL activity in thefinished products is assayed with the method described in example 4against an external calibration range of MGL in aqueous solution. Theseresults, combined with explanatory studies, show that MGL activity inthe finished products increases with the amount of enzyme introducedinto the method and that it is easily possible to encapsulate up to 32IU of MGL per ml of finished product while maintaining good stability.

In another study, three murine finished products RC-MGL-PLP1,RC-MGL-PLP2 and RC-MGL-PLP3 were prepared according to the followingmethods:

-   -   RC-MGL-PLP1: co-encapsulation of MGL and of PLP from a        suspension containing 3 mg/ml of MGL and ˜30 μM of PLP. The        final product was taken up in SAG-Mannitol, 6% BSA supplemented        with final 10 μM PLP.    -   RC-MGL-PLP2: co-encapsulation of MGL and of PLP from a        suspension containing 3 mg/ml of MGL and ˜30 μM of PLP. The        finished product was taken up in SAG-Mannitol 6% BSA.    -   RC-MGL-PLP3: this product stems from a co-encapsulation of MGL        and PLP from a suspension containing 3 mg/ml of MGL and ˜124 μM        of PLP. The final product was taken up in SAG-Mannitol 6% BSA.

In a third study, a murine finished product RC-MGL-PLP4 was preparedfrom a new batch of MGL according to the following methods:

RC-MGL-PLP4: co-encapsulation of MGL and the PLP from a suspensioncontaining 5 mg/ml of MGL and ˜35 μM of PLP. The finished product wastaken up in SAG-Mannitol 6% BSA.

Finally in a fourth study, a murine product RC-MGL-PLP5 was preparedfrom a third batch of MGL according to the following methods:

-   -   RC-MGL-PLP5: co-encapsulation of MGL and PLP from a suspension        containing 6 mg/ml of MGL and ˜100 μM of PLP. The finished        product was taken up in SAG-Mannitol 6% BSA.

The hematologic and biochemical characteristics of the three finishedproducts at D0 (after their preparation) are detailed in the tablebelow. The encapsulation yields are satisfactory and vary from 18.6% to30.5%.

RC- RC- RC- RC- RC- MGL- MGL- MGL- MGL- MGL- PLP1 PLP2 PLP3 PLP4 PLP5Hema- Hematocrit (%) 50.0 49.6 50.0 50.0 50.0 tological Corpuscle volume(fl) 46.3 46.5 46.8 42.4 45.6 data Corpuscle hemoglobin (g/dl) 24.7 24.024.2 27.4 25.1 RC concentration (10⁶/μl) 6.5 6.9 6.6 7.2 6.0 Totalhemoglobin (g/dl) 14.8 15.4 15.0 16.6 13.8 Extracellular Hb (g/dl) 0.10.1 0.1 0.2 0.05 mgl Intra-erythrocyte concentration 0.97 0.94 0.79 1.011.36 of MGL (mg/ml of RC) Intra-erythrocyte activity of MGL 12.8 12.48.8 5.0 8.6 (IU/ml of RC)* Extracellular activity (%) 0.92% 0.97% 1.32%1.18% 2.23% Intracellular activity (%) 99.08%  99.03%  98.68%  98.82% 97.77%  Encapsulation yield of MGL (%) 18.6% 30.5% 22.6% 19.4% 22.7% PLPIntra-erythrocyte concentration ND 13.4 71.4 10.2 ND of PLP (μmol/l ofRC) Intracellular PLP fraction (%) ND 99.5 98.7 98.1 ND ExtracellularPLP fraction (%) ND 0.5 1.3 1.92 ND PLP encapsulation yield (%) ND 44.857.4 30.7 ND *Calculated from the specific activity of each batch.

EXAMPLE 3. PRODUCTION OF HUMAN RCS ENCAPSULATING METHIONINE GAMMA LYASEAND PLP ACCORDING TO THE INDUSTRIAL METHOD

A pouch of leukocyte-depleted human RCs (provided by the “EtablissementFrançais du Sang”) is subject to a cycle of three washes with 0.9% NaCl(washer Cobe 2991). The freeze-dried MGL is re-suspended with 0.7% NaCland added to the erythrocyte suspension in order to obtain a finalsuspension with a hematocrit of 70%, containing 3 mg/ml of MGL and ˜30μM of PLP (stemming from the formulation of MGL). The suspension ishomogenized and it is proceeded with encapsulation according to themethod described in EP 1 773 452. The suspension from the resealing isthen incubated for 3 h at room temperature in order to remove the mostfragile RCs. The suspension is washed three times with a 0.9% NaCl, 0.2%glucose solution (washer Cobe 2991) and then re-suspended with 80 ml ofpreservation solution (AS-3). The encapsulated MGL level is assayed likein Example 4.

J0 J1 J7 Hematocrit (%) 52.0 51.6 52.7 Corpuscle volume (fl) 91.0 92.088.0 Corpuscle hemoglobin (g/dl) 30.3 29.8 31.6 RC concentration(10⁶/μl) 6.00 5.92 5.98 Total hemoglobin (g/dl) 16.4 16.2 16.6Extracellular Hb (g/dl) 0.119 0.197 0.280 Osmotic fragility (g/l) 1.17Hemolysis (%)  0.7%  1.2% 1.7% Total MGL concentration (mg/ml) 0.36 0.35MGL supernatant concentration (mg/ml) 0.01 0.01 MGL intra-erythrocyteconcentration (mg/ml, 0.68 0.67 100% Ht) Extracellular activity (%) 1.3%  1.4% Intracellular activity (%) 98.7% 98.6% Encapsulation yield(%) 19.7%

EXAMPLE 4. ASSAY OF ENCAPSULATED MGL IN THE RCS

The assay of the MGL activity in cell suspensions (total RCs) and in thesupernatants is based on a measurement of NH₃ produced by MGL. The NH₃ions are assayed indirectly by enzymatic action of glutamatedehydrogenase (GLDH) according to the kit marketed by Roche Diagnostics(11877984).

Preparation of the standards: MGL standards at different concentrationswere prepared in matrices (total or supernatant RCs) or in an aqueoussolution.

-   -   For standards in an aqueous solution, MGL is prepared at        concentrations varying from 0 to 12 μg/ml in the presence of 20        μM PLP in a phosphate buffer 100 mM at a pH of 7.2.    -   For total RC matrix standards, 10 μl of RC-LR are lysed with 90        μl of a solution containing 260 μM of PLP and of MGL at        concentrations varying from 0 to 100 μg/ml. The “total RC”        standards are then diluted 20 times with phosphate buffer 100        mM, pH 7.2.    -   For supernatant matrix standards, 10 μl of supernatants of RC-LR        are lysed with 50 μl of a solution containing 6.4 μM of PLP and        of MGL at concentrations varying from 0 to 20 μg/ml.

Pre-treatment of the samples: the samples to be assayed (10 μl) arepre-treated in the same way as the standards (addition of PLP andidentical dilutions but without addition of MGL).

Assay of MGL: 7.5 μl of standards (STD) or of samples are introducedinto the wells of a UV plate. 94 μl of reagent R1 (Roche kit) and 56 μlof reagent R2 (Roche kit) containing α-ketoglutarate in a buffersolution, NADPH and GLDH are added in order to remove the endogenous NH₃ions of the samples. After 10 min of incubation, 75 μl of L-methionineat 78.3 mM are introduced and the reaction mixtures are incubated for 30min. Degradation of NADPH into NADP⁺ is continuously tracked bymeasuring the optical density at 340 nm. For the standards and thesamples, the value of ΔOD/min is calculated over the linear domain ofthe O.D. curves obtained at 340 nm. A calibration curve ΔOD/min=f (MGLconcentration or activity in the standards) is then plotted. Theregression parameters allow determination of the MGL concentration inthe samples. This result may be expressed in mg/ml or in IU/ml (thespecific MGL activity being evaluated for each batch). Theintra-erythrocyte MGL level is obtained by a calculation with thefollowing formula:[MGL]_(intra-erythrocyte)=([MGL]_(total)−([MGL]_(supernatants)×(1-hematocrit/100))/(hematocrit/100).

EXAMPLE 5. ASSAY OF PLP IN BLOOD SAMPLES BY A HPLC METHOD

The assay of PLP in cell suspensions (total RCs) and in the supernatantsis an adaptation of the method described by Van de Kamp et al. NutritionResearch 15, 415-422, 1995. The assay is carried out with RP-HPLC(Shimadzu UFLC) with detection by fluorimetry (RF-10AXL instrument,excitation: 300 nm, emission: 400 nm). The PLP contained in the samplesis extracted with trichloro-acetic acid (TCA) at a final 6%. Aftercentrifugation (15,000×g, 10 min), the supernatants are collected andthen diluted in a mobile phase A. A 50 μl sample volume is injected on a5 μC-18 Gemini column, 250×4.6 mm (Phenomenex). The mobile phase Aconsists of 33 mM of monobasic potassium phosphate, of 8 mM of sodium1-octanesulfonate supplemented with sodium bisulfite (0.5 g/l) forintensifying the signal of the PLP and of the mobile phase B, of 33 mMof monobasic potassium phosphate and of 17% (v:v) of 2-propanol. Thegradient used is the mobile phase A (100%) with increasing proportionsof mobile phase B: an increase from 0% to 8% of B over a period of 8min. The flow through the column is maintained at 1 ml/min. The PLPconcentration in the samples is determined with an external standardrange of PLPs subject to the same TCA treatment as the samples. Theretention time of PLP is ˜3.4 min. The intra-erythrocyte PLP level isobtained by calculation with the following formula:[PLP]_(intra-erythrocyte)=([PLP]_(total)−([PLP]_(supernatants)×(1-hematocrit/100))/(hematocrit/100).

EXAMPLE 6. INCREASE IN THE PLP LEVEL IN RCS BY CO-ENCAPSULATION OF PLPWITH MGL

Suspensions of murine RCs are subject to the method for encapsulatingMGL and PLP as described in Example 2. The assay of the intracellularPLP is carried out according to the method described in Example 5.

A suspension of human RCs is subject to the method for encapsulating MGLand PLP as described in Example 3. Before the incubation step at roomtemperature, a portion of the human RC-MGL-PLPs is sampled in order tocarry out an assay of the intracellular PLP according to the methoddescribed in Example 5.

The following table compares the physiological levels of PLP in human ormurine erythrocytes with the level attained by co-encapsulation of thelatter with MGL.

Human RCs 0.11 μM Murine RCs (Natta & ~2.4 μM* Physiological level ofPLP Reynolds) (Fonda) PLP level Conditions before ~3.90 μM ~13.4 μM inRC-MGL-PLP1s or dialysis: RC-MGL-PLP2s 3 mg/ml MGL  ~30 μM PLP PLP levelConditions before ~71.4 μM in RC-MGL-PLP3s dialysis: 3 mg/ml MGL ~125 μMPLP *detail of the calculation: 7.5 nmol/g Hb ~2.4 μM (by assuming aCCMH of 32 g/dl).

EXAMPLE 7. DEMONSTRATION OF THE INCREASE IN THE PLP CONCENTRATION IN THERC-MGLS BY INCUBATION IN VITRO WITH PYRIDOXINE

A suspension of human RCs is subject to the method for encapsulating MGLas described in Example 3. Before the 3 h incubation step, a portion ofthe RCs is sampled and separated into three for volume-volume incubationwith pyridoxine at different concentrations (0 mM, 2 mM and 4 mM). Afterhomogenization, these suspensions are incubated at room temperature(RT). After 3 h and 24 h of incubation, samples of the cell suspensionsand of the supernatants (obtained after centrifugation of thesuspensions at 1000×g, at 4° C., for 10 min) are prepared and frozen fora measurement of the PLP concentration by HPLC as described in Example5.

The obtained results are shown in FIGS. 2 and 3.

In the absence of pyridoxine, the intra-erythrocyte PLP level is 3.9 μM(PLP stemming from the co-encapsulation of MGL and PLP). This PLPconcentration remains constant after 3 h and 24 h of incubation. Aslight decrease in the PLP concentration is observed at 24 h and isconcomitant with occurrence of extracellular PLP which may be explainedby hemolysis at the end of the incubation.

In the presence of pyridoxine (at 2 mM or at 4 mM), the RC-MGLs areenriched in PLP with intra-erythrocyte concentrations increased by afactor 2 after 3 h of incubation (˜8 μM of PLP) and by almost a factor 3after 24 h of incubation with occurrence of a dose effect (11 μM and 14μM for respective pyridoxine concentrations of 2 mM and 4 mM). Theseresults show that an incubation of a RC suspension encapsulating a PLPenzyme dependent on PLP with pyridoxine (PN) is capable of increasingthe intracellular PLP level in a long lasting way.

EXAMPLE 8. PHARMACOKINETICS OF RC ENCAPSULATING MGL-PLP IN MICE

The murine products RC-MGL-PLP2, RC-MGL-PLP3, RC-MGL-PLP4 andRC-MGL-PLP5 are labeled with CFSE (fluorescent) and injectedintravenously into CD1 mice. After various times (D0+15 min, D1, D2, D5,for the three products with additionally D14 and D28 for the RC-MGL-PLP4and D14 for the RC-MGL-PLP5 product), the mice are sacrificed and theblood is collected on a lithium heparinate tube kept at +4° C. away fromlight for determining the pharmacokinetics. The proportion of red bloodcells labeled with CFSE in the whole blood is determined by a flowcytometry method. Five microliters of whole blood are diluted in 1 ml ofPBS 0.5% BSA and each sample is passed in triplicate (counting of 10,000cells in FL-1; cytometer FC500, Beckman Coulter). The evaluation of thesurvival of red blood cells loaded with MGL is obtained by adding theproportion of RCs labeled with CFSE at different times to the proportionof RCs labeled with CFSE at T0+15 min (100% control). The differentobtained percentages for each time are copied onto a graph (FIG. 4)illustrating the proportion of RCs loaded with MGL in circulation versustime.

The determination of the proportion of RCs marked with CFSE incirculating blood at different times shows its excellent stability ofthe four products in vivo in mice, up to 120 h post-injection(83.5±0.6%, 94.7±0.6%, 87.3±5.6% and 76.8±1.3% survival rate,respectively). For the product RC-MGL-PLP4, the pharmacokinetic studyover 29 days showed that the half-life of the red blood cellsencapsulating MGL is ˜12.6 days.

EXAMPLE 9. L-METHIONINE DEPLETION AT 24 H

The murine products RC-MGL-PLP1, RC-MGL-PLP2 and RC-MGL-PLP3 preparedand characterized as in Example 2 are injected intravenously to CD1 miceat a dose of 8 ml/kg. After 6 h, ˜0.09 mg of pyridoxine (i.e. 150 μL ofa 2.9 mM pyridoxine hydrochloride solution) were injected to micereceiving RC-MGL-PLP2. The L-Met plasma level was evaluated at 24 h byHPLC-MS-MS (Piraud M. et al., Rapid Commun. Mass Spectrum. 19, 3287-97,2005). The following table shows the depletions obtained in the variousgroups of injected mice.

L-Met Administered Methods for providing plasma % of product the PLPco-enzyme level (μM) depletion — Feeding 82.7 ± 22.5 — RC-MGL- Feeding46.3 ± 3.5  44% PLP1 PLP in the finished product (~5 μmol/l, RC) PLP inthe preservation solution of the finished product (10 μM) RC-MGL-Feeding 22.3 ± 4.9  73% PLP2 + PLP encapsulated in the finishedpyridoxine product (~13.4 μmol/l RC) IV injection of ~0.09 mg ofpyridoxine RC-MGL- Feeding 29.7 ± 4.6  64% PLP3 PLP encapsulated in thefinished product (~71.4 μmol/l RC)

The L-Met plasma level was evaluated to be 82.7±22.5 μM in control mice.The product RC-MGL-PLP1 containing encapsulated MGL with a low PLPconcentration leads to 44% depletion of L-Met, 24 h after administrationof the product. We put forward the assumption that the PLP added intothe preservation solution of the product is not available for the enzymeMGL since 1) it is in majority bound to the BSA present in thepreservation solution and 2) it cannot pass through the membrane of theRC.

The results show that a more consequent provision of PLP in the redcorpuscle either by IV injection of pyridoxine (RC-MGL-PLP2) or byencapsulation of PLP at a stronger concentration gives the possibilityof obtaining L-Met depletions ˜1.5 times greater (73% and 64% depletionrespectively).

EXAMPLE 10. PHARMACODYNAMICS OF RC-MGLS

The MGL enzyme in its free form is injected intravenously to CD1 mice ata dose of 8 ml/kg. Two series of injections were made, the first with anenzyme concentration at 0.45 mg/ml (product MGL-L1), the second at atwice higher concentration (0.90 mg/ml; product MGL-L2). Six hours afterinjection, ˜0.09 mg of pyridoxine (i.e. 150 μL of a 2.9 mM pyridoxinehydrochloride solution) are injected into mice receiving MGL-L1 andMGL-L2. The L-Met plasma level is evaluated by HPLC-MS-MS at 15 min, 24h, 48 h, and 120 h post-injection of MGL-L1 and at 15 min, 24 h, 48 h,120 h and 144 h post-injection of MGL-L2. FIG. 5 shows the depletionsobtained in the various groups of injected mice.

The results show that in both experimental groups, a very strongL-methionine depletion (≈4 μM) and rapid overtime (15 minpost-injection). However, this depletion is transient and not maintainedover time, the L-methionine levels returning into the control values 24h after injection, and this in spite of the initial provision of P5P(present in the dilution buffer but also in the formulation of theenzyme taken up in water) and the supplementation with vitamin B6identical with the one carried out for the RC-MGL-PLP2 product. Theactivity of free MGL is therefore lost between 15 min and 24 hpost-injection, probably due to rapid removal of the circulating enzyme.

In a second phase, the murine products RC-MGL-PLP2 and RC-MGL-PLP3prepared and characterized as in Example 2 are injected intravenously toCD1 mice at a dose of 8 ml/kg. After 6 h, ˜0.09 mg of pyridoxine (i.e.150 μL of a 2.9 mM pyridoxine hydrochloride solution) are injected intothe mice receiving RC-MGL-PLP2. The L-Met plasma level is evaluated tobe at 15 min, 24 h, 48 h, and 120 h post-injection by HPLC-MS-MS. FIG. 6and the following table show the depletion obtained in the variousgroups of injected mice.

The results show that in both experimental groups, an L-methioninedepletion stabilized at ˜35 μM and maintained over time (from 48 to 120h post injection). These results indicate that supplementation with PLPor with its precursor (vitamin B6) gives the possibility of maintainingan activity of the MGL encapsulated in RCs for at least 120 h afterinjection in mice. As an indication, the L-methionine concentrations inplasma 24 and 120 h post-injection for the various products (free formof MGL or co-encapsulated in red blood cells with PLP) are given in thefollowing table:

L-methionine level (μM) At T0 (ctrl) At 24 h At 120 h Free MGL MGL-L168.2 ± 21.7 57.0 ± 8.0  62.5 ± 12.0 MGL-L2 68.2 ± 21.7 57.3 ± 5.1 51.0 ±8.5 Encapsulated RC-MGL-PLP2 82.7 ± 22.5 29.7 ± 4.6 36.0 ± 2.6 MGLRC-MGL-PLP3 82.7 ± 22.5 22.3 ± 4.9 34.3 ± 7.4

In order to assess the pharmacodynamics over times of more than 120 h,the murine product RC-MGL-PLP4 having a concentration of encapsulatedMGL of 0.5 mg/ml of enzyme in the finished product is injectedintravenously to CD1 mice with a dose of 10 ml/kg. After 6 h, ˜0.09 mgof pyridoxine (i.e. 150 μL of a 2.9 mM pyridoxine hydrochloridesolution) are injected into the mice receiving RC-MGL-PLP4. The L-Metplasma level is evaluated by HPLC-MS-MS. FIG. 7 shows the depletionsobtained at 15 min, on D1, D2, D5, D14 and D28 after injection.

The results show a significant L-methionine depletion (≈10 μM against≈68 μM for the control) and rapid depletion over time (15 minpost-injection). However, this depletion is slightly stabilized between24 h and 48 h to a value of ˜25 μM and increases up to ˜40 μM after 5days so as to finally attain the control values at about 12 days afterinjection of RC-MGL-PLP4.

Finally, the residual activity of the injected MGL enzyme is determinedaccording to the assay method described in Example 4 in the presence ofPLP. FIG. 8 hereafter lists the residual activities versus time for thefree enzyme MGL-L2, the various products RC-MGL-PLP and for the TPLenzyme (data from the literature).

The results show that by encapsulating MGL in murine red blood cells itis possible to retain strong enzymatic activity at 24 h (residualactivity comprised between ˜60 and 100%). This residual activityslightly decreases at 48 h (˜35 to 100%) and is maintained up to 120 h,i.e. 5 days after injection, at values comprised between ˜20 and ˜65%.The residual activity of the MGL in its free form drastically drops inthe first minutes post-injection so as to be almost zero at 24 h(residual activity<10%). By comparison, the residual activity of the TPLinjected in its free form is copied on the graph and 5 h afterinjection, the latter is only at most 37% (Elmer et al., 1978). Themeasurement of the residual activity clearly shows the benefit ofencapsulation of the PLP enzymes in red blood cells for maintainingtheir enzymatic activity.

What is claimed is:
 1. One or more erythrocyte(s) encapsulating aplurality of pyridoxal phosphate (PLP)-dependent enzyme molecules, theerythrocytes comprising a sufficient amount of pyridoxine kinase(PN-kinase) and pyridoxine phosphate oxidase (PNP oxidase) to produce asufficient amount of PLP from PLP precursor present in a subject'sbloodstream to maintain a sufficient portion of theerythrocyte-encapsulated PLP-dependent enzyme molecules in theirholoenzyme forms to preserve enzymatic activity beyond 24 hours afterinjection or infusion into the subject in need of said enzymaticactivity.
 2. The erythrocyte(s) of claim 1, wherein the PLP-dependentactivity persists in the subject for at least 1-15 days after injectionor infusion.
 3. The erythrocyte(s) of claim 1, wherein the PLP-dependentactivity persists in the subject for at least 15 days after injection orinfusion as measured by a depletion percentage of more than 20-50% ofthe PLP-dependent enzyme's substrate in the plasma of the patient orsubject.
 4. A pharmaceutical composition comprising the erythrocytes ofclaim 1 and a pharmaceutically acceptable vehicle.
 5. The composition ofclaim 4, further comprising a non-phosphate precursor of PLPencapsulated in the erythrocytes and/or present outside theerythrocytes.
 6. The composition of claim 5, wherein the non-phosphateprecursor is selected from pyridoxal, pyridoxine, pyridoxamine, andcombinations thereof.
 7. The composition of claim 4, further comprisinga phosphate PLP precursor, encapsulated in the erythrocytes.
 8. Thecomposition of claim 7, wherein the phosphate precursor is selected fromthe group consisting of pyridoxine phosphate (PNP), pyridoxaminephosphate (PMP), and combinations thereof.
 9. The composition of claim4, comprising from about 0.01 to about 30 mg of PLP-dependent enzyme perml of erythrocytes.
 10. The composition of claim 7, comprising fromabout 0.01 to about 30 mg of PLP-dependent enzyme per ml oferythrocytes.
 11. The composition of claim 4, comprising from about 0.05to 600 μmol of encapsulated PLP and/or pyridoxine phosphate (PNP) and/orpyridoxamine phosphate (PMP), per liter (L) of erythrocytes.
 12. Thecomposition of claim 11, comprising from about 5 to about 50 μmol ofencapsulated PLP and/or PNP and/or PMP, per liter (L) of erythrocytes.13. The composition of claim 4, further comprising pyridoxine kinase(PN-kinase), pyridoxine phosphate oxidase (PNP-oxidase), and an agentinhibiting pyridoxal phosphate phosphatase (PLP-phosphatase).
 14. Thecomposition of claim 4, wherein the PLP-dependent enzyme comprises amethioninase, a tyrosine phenol-lyase, a tyrosine aminotransferase or acystathionine beta-synthase.
 15. The composition of claim 7, wherein thePLP-dependent enzyme comprises a methioninase, a tyrosine phenol-lyase,a tyrosine aminotransferase or a cystathionine beta-synthase.
 16. Thecomposition of claim 4, comprising from about 0.05 to about 10 mg ofPLP-dependent enzyme per ml of erythrocytes.
 17. The composition ofclaim 7, comprising from about 0.05 to about 10 mg of PLP-dependentenzyme per ml of erythrocytes.
 18. The composition of claim 4,comprising from about 0.5 to about 100 μmol of encapsulated PLP and/orpyridoxine phosphate (PNP) and/or pyridoxamine phosphate (PMP), perliter (L) of erythrocytes.
 19. The composition of claim 4, comprisingfrom about 5 to about 50 μmol of encapsulated PLP and/or pyridoxinephosphate (PNP) and/or pyridoxamine phosphate (PMP), per liter (L) oferythrocytes.